Biomedical subjects

Clive R Taylor

Publications and source records attributed to Clive R Taylor.

21 records · Page 2Linked to original sources

PubMed2002

DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: heating under the influence of pH.

During the course of diagnostic surgical pathology, pathologists have established a large collection of formalin-fixed, paraffin-embedded tissues that form invaluable resources for translational studies of cancer and a variety of other diseases. Accessibility of macromolecules in the fixed tissue specimens is a critical issue as exemplified by heat-induced antigen retrieval (AR) immunohistochemical (IHC) staining. On the basis of observations that heating may also enhance in situ hybridization (ISH) and the similarity of formalin-induced chemical modifications that occur in protein and in DNA, we designed a study to examine the efficiency of DNA extraction from archival formalin-fixed, paraffin-embedded tissues using an adaptation of the basic principles of the AR technique, i.e., heating the tissue under the influence of different pH values. Archival paraffin blocks of lymph nodes, tonsil, and colon were randomly selected. Each paraffin block was prepared in 34 microtubes. For each paraffin block, one tube was used as a control sample, using a non-heating DNA extraction protocol. The other 33 tubes were tested using a heating protocol under 11 variable pH values (pH 2 to 12) under three different heating conditions (80, 100, and 120C). Evaluation of the results of DNA extraction was carried out by measuring yields by photometry and PCR amplification, as well as kinetic thermocycling (KTC)-PCR methods. In general, lower pH (acid) solutions gave inferior results to solutions at higher pH (alkaline). Heating tissues at a higher temperature and at pH 6-9 gave higher yields of DNA. There appeared to be a peak in terms of highest efficiency of extracted DNA at around pH 9. The average ratios 260:280 of extracted DNA also showed better values for samples heated at 120C. PCR products of three primers showed satisfactory results for DNA extracted from archival paraffin-embedded tissues by heating protocols at pH 6-12, with results that were comparable to the control sample subjected to the standard non-heating, enzymatic DNA extraction method. This study is the first to document the use of heating at an alkaline pH for DNA extraction from archival formalin-fixed, paraffin-embedded tissues, a recommendation based on the principles of AR for protein IHC. These findings may lead to a more effective protocol for DNA extraction from archival paraffin-embedded tissues and may also provide enhanced understanding of changes that occur during formalin-induced modification of nucleic acids.

Antigens↗

PubMedDate not supplied

LEA.135 expression: identifies low-risk patients with breast ductal carcinoma in situ.

The expression of a cell surface-associated sialoglycoprotein (LEA.135), which has been shown to be significantly associated with decreased incidence of recurrence and increased overall survival of patients with primary invasive breast carcinoma, was evaluated in a retrospective study to identify subsets of patients with breast ductal carcinoma in situ (DCIS) who are at high risk of subsequently developing invasive breast carcinoma. The study was carried out by an immuno-histochemical method on formalin-fixed and paraffin-embedded tissue sections from 63 patients initially with DCIS. Pathological parameters such as DCIS histological type, nuclear grade, as well as time and type of recurrence (either a second DCIS or the diagnosis of locally invasive breast carcinoma) together with follow-up in years were available for the cohort of patients. A comparison of recurrence was made of patients whose tumor cells exhibited LEA.135 expression (24 +/- 8% recurring by 7 years), compared with those patients whose specimens showed the absence of LEA.135 expression (41 +/- 10% recurring by 7 years). A statistically significant univariant association between LEA.135 expression and the absence of recurrence of DCIS or development of locally invasive breast carcinoma was observed, suggesting a favorable prognostic significance of LEA. 135 expression (log-rank p = 0.032). It is worthy of mention that none of the LEA. 135-positive patients developed recurrence as DCIS or locally invasive breast carcinoma (0.24 +/- 0.08) after 5 years of the initial diagnosis of DCIS, whereas those from LEA. 135-negative progressively increased their recurrence at 5 years (0.30 +/- 0.09), 7 years (0.41 +/- 0.10) and 10 years (0.63 +/- 0.12). The results of this pilot study show that LEA.135 expression is significantly associated with a favorable prognosis of patients with DCIS, leading to a decreased incidence of recurrence.

Breast Neoplasms↗

PubMedDate not supplied

LEA-135 expression: its association with a lower risk of recurrence and increased overall survival of patients with lymph node-positive primary invasive breast cancer.

A retrospective study was undertaken to determine and compare the prognostic significance of LEA-135 protein expression by immunohistochemistry with other prognostic pathological parameters, with respect to recurrence and overall survival. This study was conducted in freshly-frozen tissue sections from a cohort of 367 patients having primary invasive breast cancer, with axillary lymph node metastasis. The association of LEA-135 expression was compared with estrogen and progesterone receptor status, segmentectomy or radical mastectomy and hormonal therapy or chemotherapy in terms of recurrence or disease-free survival. Pathologic parameters including tumor size, histological tumor type and histological grade, as well as age of patients at the time of initial diagnosis, and the treatments, together with a median follow-up of 8.8 years were contemplated for the study. Among these parameters, tumor size and histological grade were individually and significantly associated with an increased probability of recurrence (log rank p<0.001 in both cases) and short survival (log ranks p<0.001 and p=0.002, respectively), whereas age was only significantly associated with an increased probability of recurrence (log rank p=0.002) by univariate analysis. By multivariate analysis, both tumor size and histological grade remained statistically significant for recurrence (log rank p<0.001 and p=0.013, respectively) and overall survival (log ranks p<0.001 and p=0.016, respectively). Among the prognostic biomarkers, both ER and PR expression were associated with a decreased rate of recurrence (log ranks p<0.001 and p=0.008, respectively) and overall survival (log ranks p<0.001 and p=0.002, respectively) by univariate analysis. By multivariate analysis, only the ER expression remained significantly associated with a decreased recurrence and increased overall survival (log ranks p=0.023 and p=0.002, respectively). Patients with high (>50% positive cells) or moderate (5-50% positive cells) number of LEA-135-positive cells had a lower probability (46%) of recurrence at 10 years after surgery compared to 76% in LEA-135-negative patients (log rank p<0.001) by univariate analysis. Moreover, the probability of overall survival was higher in patients with high or moderate expression of LEA-135 (46% and 47%, respectively) compared to LEA-135-negative patients (24%) by univariate analysis (log rank p=0.009). By multivariate analysis, the association remained statistically significant for recurrence (log rank p<0.001) and survival (log rank p=0.002). However, there was no significant association between LEA-135 and any of the pathological parameters, age, hormone receptor status, the mode of surgery or the form of therapy (chemo- and/or hormonal) received by this cohort of patients. The results show that an improved prognosis was directly associated with the density of LEA-135-positive cancer cells, while loss of LEA-135 expression was associated with an aggressive phenotype of cancer cells during breast cancer progression. Thus, LEA-135 expression can be implicated as a significant and independent biomarker to identify and distinguish high- from low-risk patients with lymph node-positive invasive breast cancer for an aggressive treatment. Moreover, according to the present results, LEA-135 expression appears to be associated with the tumor cells that have retained certain normal biological characteristics, leading to their lack of aggressiveness and hence a better prognosis.

Age Factors↗